Mutation and nuclear stage in Schizosaccharomyces pombe I. An experimental approach to the role of recombination in mutation induction

A reverse mutation system using G1 and G2 cells of Schizosaccharomyces pombe is described. In order to enable the system to deal with the problem of mutation dependence on recombination, tests were performed on (i) the homogeneity of cell populations with respect to nuclear stage; (ii) the fate of cells during post-irradiation incubation; (iii) the colony-forming ability of G1 and G2 revertants, and (iv) cell viability on the mutation plates. On the basis of the results, it is thought that, using this system, information can be obtained on the role of recombinational events in the process of mutation induction.     

Characterization of chemically induced mutations in the ad-I locus of Schizosaccharomyces pombe

An attempt to assess the frequencies of mutations of the base-pair substitution type and of the addition/deletion type was undertaken in 64 ICR-170-, 28 MNNG- and 50 EMS-induced ad-1 mutant strains of Schizosaccharomyces pombe.By using temperature sensitivity, osmotic remediability, and interallelic complementation, sensitivity to nonsense suppressors and revertibility tests with 2-methoxy- 6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) as diagnostic criteria to distinguish between the two types of alterations, the following conclusions were reached: (1) The mutational alteration in all of the MNNG-induced and in at least 74% of the ethyl methanesulfonate(EMS)-induced mutant strains is of the base-pair substitution type; (2) Both types of mutation were found amongst ICR-170-induced strains.     

Serum effect on the yield of chemically induced 8-azaguanine-resistant mutants in Chinese hamster cell cultures

Induction of 8-azaguanine-resistant mutants by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was studied in Chinese hamster cell cultures. The rate of expression of newly induced mutations and the total yield of mutants were affected by the fetal calf serum used for the growth medium. A correlation was observed between reduced growth rate of cells, reduced expression rate and low yield of mutants.The involvement of a repair process is discussed.     

Response of Escherichia coli to ethyl methanesulfonate: Influence of growth phase and repair ability on survival and mutagenesis

The effects of altering the cell growth rate (physiological state) and DNA repair capacity (genetic state) on susceptibility to inactivation and mutagenesis by ethyl methanesulfonate (EMS) were studied in 4 strains of E. coli. Logarithmic and stationary phase cells of the polymerase I deficient mutant, P₃₄₇8 polA, a recombination deficient mutant, DZ₄₁₇ recA, and the respective parental strains, W₃₁₁₀pol⁺ and AB₂₅₃ rec⁺, were exposed to EMS and the surviving fraction and mutant frequency determined. At the same EMS concentration both mutants were more susceptible to inactivation than the parental strains. In all 4 strains, log phase cells were more sensitive to inactivation than stationary cells. The surviving fraction of stationary cells exceeded log cells by a factor of 18 for polA, 6 for recA, and about 2 for the parental strains. In all strains, except recA, log phase cells exhibited higher spontaneous mutant frequencies than stationary phase cells. At the same concentration of EMS, survivors of both polA and recA showed more than 10-fold higher induced frequencies than the wild types. However, at the same survival levels the repair deficient mutants exhibited induced mutant frequencies comparable to the repair proficient strains. There was no significant effect of growth phase on EMS induced mutability in recA or the parental strains. In marked contrast, the polymerase I deficient mutant shows both a higher spontaneous frequency and a greater than 10-fold higher EMS induced mutant frequency in log phase cultures compared to stationary phase cultures. Our results support the hypothesis that cellular susceptibility to alkylating agents is influenced by both the genetic capability for repair and the particular physiological state of the cell.     

A yeast strain for simultaneous detection of induced mitotic crossing over,mitotic gene conversion and reverse mutation

A diploid yeast strain is described which can be used to study induction of mitotic crossing over, mitotic gene conversion and reverse mutation.Mitotic crossing over can be detected visually as pink and red twin sectored colonies which are due to the formation of homozygous cells of the genotype ade240/ade240 (deep red) and ade-2-119/ade2-119 (pink) from the originally heteroallelic condition ade2-40/ade2-119 which forms white colonies.Mitotic gene conversion is monitored by the appearance of tryptophan non-requiring colonies on selective media. The alleles involved are tryp5-12 and trp5-27 derived from the widely used strain D4.Mutation induction can be followed by the appearance of isoleucine non-requiring colonies on selective media. D7 is homoallelic ilv1-92/ilv1-92. The isoleucine requirement caused by ilv1-92 can be alleviated by true reverse mutation and allele non-specific suppressor mutation.The effects of ethyl methanesulfonate (EMS), nitrous acid, ultraviolet light and hycanthone methanesulfonate were studied with D7 stationary phase cells. Mitotic crossing over as monitored by red/pink twin sectored colonies was almost equally frequent among normal and convertant cells. This showed again that mitotic recombination is not due to the presence fo a few cells committed to meiosis in an otherwise mitotic cell population.The dose-response curves for induction of mitotic gene conversion and reversion of the isoleucine requirement were exponential. In contrast to this, the dose-response curve for induction of twin sectored red and pink colonies reached a plateau at doses giving about 30% cell killing. This could partly be due to lethal segregation in the progeny of treated cells.None of the agents tested would induce only one type of mitotic recombination, gene conversion or crossing over. There was, however, some mutagen specificity in the induction of isoleucine prototrophs.     

An evaluation of Tween 80 effects on the survival and DNA repair in Escherichia coli following UV or gamma irradiation.

The notion that Tween 80 may be a DNA-repair inhibitor was tested with Escherichia coli. The results indicate that cell growth, colony-forming ability, and the rate and extent of removal of thymine-containing dimers from DNA are unchanged in the presence of Tween 80. We conclude that this detergent does not increase or diminish the effect of UV or gamma irradiation to bacteria.     

Genetic analysis of X-linked mutagen-sensitive mutants of Drosophila melanogaster

Mutants at 2 new loci which control mutagen-sensitivity are described. Mutants at both loci are female-sterile and are hypersensitive to killing by MMS; neither increases the frequency of sex-linked recessive lethals. A screen of previously described female-sterile and meotic mutants has revealed that a number of these are also sensitive to mutagens. In addition, several new mutants have been identified on the basis of sensitivity to either HN2 or MMS. An anlysis of complementation data suggests that all of the X-linked genes controlling sensitivity to MMS may now have been identified. Among the new mei-41 alleles are mutants which show verly little meiotic nondisjunction or loss. Cytogenetic mapping of previously known mutants is also described. The mutants mus(1)104^D1 and mei-41^D5 are located in th eregion 14B13±?14D1,2 on the polytene chromosome map, and they map very close to each other genetically. Cytogenetically mus(1)101^D1 is between salivary chromosome bands 12A6,7 and 12D3, mus(1)103^D1 is between bands 12A1,2 and 12A6,7, and mus(1)-109^A1 is in section 8F3-9A2.     

Isolation and characterization of temperature-sensitive mutants in a Chinese hamster cell line

A replica plating method was used for the isolation of temperature-sensitive (ts) mutants after treatment of Chinese hamster cells with ethyl methanesulfonate (EMS). No significant increase in ts mutants was found after this treatment. The limitations and advantages of the replicating procedure to detect such differences, as well as an alternative method, are discussed.Mutants isolated were classified into two general groups—density-dependent and clear-cut—as measured by survival at low and high cell densities at the restrictive temperature. The density-dependent mutants may be truly “leaky”, losing a metabolite to the medium at an excessive rate at the restrictive temperature. On the other hand, the one clear-cut mutant analyzed extensively dies at a rate determined by its ability to utilize one or more components from the medium. It shows an inverse density relationship in rate of death, as inferred from rates of macromolecular synthesis, as opposed to its growth rate at the permissive temperature.     

The effects of radiation exposure-rate and exposure fractionation on the frequencies of recessive and dominant lethals in immature oocytes of Drosophila melanogaster

A measure of seedling viability was used to estimate the homozygous geneticc load of seedlings from mutagenized populations of Oenothera. The breeding protocol forced genomes to homozygosity. Pollen from control and mutagenized Oenothera hookeri T. and G. strain Johansen, a seven-paired lethal-free stock, was used to pollinate a translocation stock with balanced lethals. The hybrid formed a complete translocation ring and upon selfing yielded two types of plants, a translocation heterozygote similar to its hybrid parent and a seven-paired plant homozygous for the 7 chromosomes obtained from Johansen. Genetic markers allowed the identification of seven-paired seedlings in the cotyledon stage. Control hybrids averaged a recovery of 57.5% seven-paired seedlings. Hybrids obtained from plants that had been mutagenized by seed treatment with 15000 R X-rays, 0.04 M ethyl methanesulfonate (EMS), and 0.08 M EMS averaged 48.3%, 19.2%, and 6.0% recovery of seven-paired forms, respectively. The data are used to estimate the genetic load and lethal equivalents in each population. The implications of these results are evaluated with reference to mutation breeding of plant populations.     

Prophage induction in Haemophilus influenzae and its relationship to mutation by chemical and physical agents

It is known that UV, X-rays, MMC and MMS are not mutagenic for H. influenzae, whereas HZ, EMS and MNNG are potent mutagens for this bacterium. All of these agents, however, are known to be both mutagenic and able to induce prophage in E. coli. We report here that all the agents except HZ induce prophage in H. influenzae, and EMS even induces in the recombination-defective recl mutant, which is non-inducible by UV, MMC, MNNG and MMS. MMS did not cause single-strand breaks or gaps in DNA synthesized after treatment of H. influenzae, but EMS and MNNG produced them. EMS caused more breaks in DNA synthesized before treatment than in that synthesized after treatment. On the other hand we did observe such breaks or gaps induced in E. coli in DNA synthesized posttreatment by EMS as well as by MMS and MNNG, at comparable survival levels.     

Mutagenesis by nitrosoguanidine, ethyl methanesulfonate, and mutator gene mutH in continuous cultures of Escherichia coli.

Induction of T5-R mutations by alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS) was examined in glucose limited chemostat cultures of non-mutator and mutator (mutH) bacteria. In agreement with the proposal that NTG mutagenizes DNA at the replication fork, this mutagen (6.8 X 10-minus 6 M) showed replication-dependent mutagenesis in continuous culture. EMS (5-10-minus M)) induced mutagenesis could not be correlated with growth rate, which probably means that induction of mutagenic lesions (promutations) by this mutagen does not involve replicating genes. A large synergic response was found for the mutH gene in combination with NTG, supporting the hypothesis that the mutH gene product acts during DNA replication.     

Genetic toxicity of procarbazine in bacteria and yeast.

Procarbazine [N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride] is used to treat Hodgkin's disease. This compound was tested in vitro without and with S10 fraction from mice liver (microsomal assay) using Saccharomyces cerevisiae strain D7, Salmonella typhimurium (strains TA98, TA100, TA1535) and in vivo in Swiss albino mice (host-mediated assay) using D7. Procarbazine, without S10 fraction, is highly toxic and induced mitotic crossover, gene conversion, and reverse mutation in D7. It had a toxic effect on all the Salmonella strains; but did not induce reverse mutations at the histidine loci. Procarbazine, with S10 fraction, was less toxic and did not induce genetic effects in yeast or Salmonella. In the host-mediated assay, no genetic effects were seen.     

Comparative analysis of deletion and base-change mutabilities of Escherichia coli B strains differing in DNA repair capacity (wild-type, uvrA-, polA-, recA-) by various mutagens.

Dose-response curves were compared for deletions [ColBR (resistant to colicin B) mutations being more than 80% deletions] and base changes (reversion of argFam to prototrophy argplus) induced in the same set of E. coli strains (wild-type for DNA repair, uvrA-, polA- and recA-) by N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), hydroxylamine (HA), 4-nitroquinoline I-oxide (4NQO), mitomycin C (MTC, UV and X-rays. All these agents induced deletions as well as base changes in the wild-type strain. Thus chemical mutagenesis differed in E. coli and bacteriophages in vitro, for HA, NTG, EMS and perhaps UV produced only point mutations in phage Tr. The patterns of deletion and base-change mutability in E. coli were surprisingly similar. (I) The recombination less recA- strain was mutable by only three (NTG, EMS, HA) of the seven mutagens for either deletions or base changes. (2) The uvrA- strain, unable to excise pyrimidine dimers, was very highly mutable by 4NQO and UV but immutable by MTC for both deletions and base changes. (3) The polA- strain, defective in DNA polymerase I due to a non-suppressible mutation, was very highly mutable by HA and highly mutable by MTC and 4NQO for both deletions and base changes but was highly mutable only for deletions by UV and X-rays, remaining normally mutable by the other agents for both deletions and base changes despite its high sensitivity to their inactivating action. We conclude that errors in the recA-dependent repair of induced DNA damage (after 4NQO, MTC, UV and X-rays) or errors in replication enhanced by damage to the replication system or to the template strands (after NTG, EMS, and HA) give rise to deletions as well as to base changes. From a comparative analysis of 14 dose-response curves for deletions and base changes, we conclude that the order of mutagenic efficiency relative to killing is (EMS, NTG) greater than (UV, 4NQO) greater than HA greater than (X-rays, MTC), and that X-rays, 4NQO, HA and MTC induce more ColBR deletions than Argplus base changes, whereas UV and EMS induce ColBR deletions and Argplus base changes at nearly equal rates and the specificity of NTG is intermediate between these two types.     

Induction of dominant-lethal mutations after administration of ethylenethiourea in combination with nitrite or of N-nitroso-ethylenethiourea in mice

Mutagenic potentials of ethylenethiourea (ETU) in combination with sodium nitrite or of N-nitroso-ETU, a nitrosttion product of ETU in vitro, were investigated in the mouse dominant-lethal test. Simultaneous 5-day p.o. administration of ETU (150 mg/kg) and sodium nitrite (50 mg/kg) caused a significant decrease in the percentage pregnancy and the number of implants in weeks 5 and 6 of testing, although no effects were obtained on these indices when the chemicals were applied separately. However, in the group treated with 30 mg ETU plus 10 mg sodium nitrite per kg no dominant-lethal mutations were induced. 5-day oral administration of 100 mg of N-nitroso-ETU per kg also exhibited similar effects to those observed after treatment with 150 mg ETU plus 50 mg sodium nitrite per kg.     

Metronidazole (Flagyl): lack of induction of sister-chromatid exchanges in CHO-K1 cells

328 X-linked recessive lethal mutations induced in late spermatids by hycanthone methanesulfonate were tested for coverage by duplications that comprised, in total, about 24% of the euchromatic X chromosome; 78 lethals appeared to be covered. Crossover localization tests of a random sample of 38 non-covered lethals revealed 4 chromosomes carrying a lethal within a duplicated segment. Lethals localized to a particular region were crossed to reference deficiencies and single-locus mutations, and inter se, to ascertain their genetic extent. The proportion of multi-locus deletions among these 78 covered and 4 non-covered lethals was 3/48, 1/10 and 13/24 for the distal, medial and proximal regions, respectively. A storage period of 9 days did not noticeably influence these proportions. In the sample of 38 non-covered lethals, and among 17 of the covered single-site lethals, 4 cases of strong crossover suppression were detected. Comparison of these results with data obtained with other mutagens suggests that induction of multi-locus deletions, and possibly of other types of chromosome rearrangement, could in part depend on other mechanisms than those acting in the formation of translocations and chromosome loss. For the purpose of mutagen testing, these findings imply that, in Drosophila, results in the regular genetic tests for chromosome breakage events do not always accurately predict the capacity of a mutagen to induce multi-locus deletions. This is of importance since transmissible multi-locus deletions have been considered a significant source of genetic damage in man.     

The induction of chromosomal structural changes in male chickens by the alkylating agents: triethylene melamine and ethyl methanesulfonate

E. coli chromosomal DNA wastreated with various Pt co-ordiantion compounds and then used as donor DNA in E. coli transformation. Genetic analysis of transformants obtained with Pt-treated DNA showed effects of cis-diamminedichloroplatinum(II) (cis-Pt(II)) and cis-dimethyl-1,3-diaminopropane CL₄ (cis-Pt(IV) (DMDAP) on the processing of DNA. With trans-diamminedichloroplatinum(II) (trans-Pt(II)) appllied in similar concentrations no effects were found.The effects of cis-Pt(II) and cis-Pt(IV) (DMDAP) on the genetic processing were different. The effects of cis-Pt(II) could be explained by assuming intra-strand crosslinks as an important lesion.     

Mitochondrial mutagenesis in Saccharomyces cerevisiae V. Ethyl methanesulfonate

Mitochondrial genomes are more sensitive to the lethal action of EMS than are nuclear genomes of S. cerevisiae. EMS induces efficiently only some types of mutation in nuclear genomes of yeast, and probably the same is true for induction of mutations non-lethal to the mitochondrial genomes.